This application proposes to do a detailed study of the phenotypic, genotypic, virologic, and biochemical characteristics of HIV-1 isolates that are dually resistant to AZT and 3TC. Susceptibility of HIV-1 to AZT and 3TC will be tested with a PBMC assay as well as a recombinant virus assay using low-passage isolates and biologically cloned virus. The nucleotide sequence of full-length molecular clones of RT from dually resistant strains will be determined and compared to AZT-sensitive strains. The association of specific mutations with AZT phenotype will be examined by a linear mixed-effects model. Candidate point mutations will be confirmed by in vitro mutagenesis and phenotypic analysis of the resulting recombinant viruses. The growth kinetics of dually resistant viruses will also be compared to wild-type and singlely resistant isolates. To characterize the biochemical properties of dually resistant RTs, the genes encoding the p66 and p51 RT subunits will be cloned separately into E. coli expression vectors and purified to near homogeneity. Mechanistic studies with reconstituted p66/p51 heterodimers will be performed using synthetic homopolymeric, as well as native heteromeric, RNA templates.